Cytosolic Phospholipase A 2 Is an Effector of Jak/STAT Signaling and Is Involved in Platelet-derived Growth Factor BB-induced Growth in Vascular Smooth Muscle Cells*

Platelet-derived growth factor-BB (PDGF-BB) is a potent mitogen and chemoattractant for vascular smooth muscle cells (VSMC). To understand its mitogenic and chemotactic signaling events, we studied the role of cytosolic phospholipase A 2 (cPLA 2 ) and the Jak/STAT pathway. PDGF-BB induced the expression and activity of cPLA 2 in a time-dependent manner in VSMC. Arachi- donyl trifluoromethyl ketone, a potent and specific inhibitor of cPLA 2 , significantly reduced PDGF-BB-in- duced arachidonic acid release and DNA synthesis. PDGF-BB stimulated tyrosine phosphorylation of Jak-2 in a time-dependent manner. In addition, PDGF-BB ac-tivated STAT-3 as determined by its tyrosine phosphorylation, DNA-binding activity, and reporter gene expression, and these responses were suppressed by AG490, a selective inhibitor of Jak-2. AG490 and a dom-inant-negative mutant of STAT-3 also attenuated PDGF-BB-induced expression of cPLA 2, arachidonic acid re- lease, and DNA synthesis in VSMC. Together, these results suggest that induction of expression of cPLA 2 and arachidonic acid release are involved in VSMC growth in response to PDGF-BB and that these events are mediated by Jak-2-dependent STAT-3 activation.

Increased vascular smooth muscle cell (VSMC) 1 growth is one of the major components in the thickening of the arterial wall in the pathogenesis of atherosclerosis and restenosis (1). A variety of molecules (including peptide growth factors, hormones, eicosanoids, and oxidants) that are generated at the site of arterial injury and/or inflammation can influence the growth and migration of VSMC (1)(2)(3)(4)(5)(6). Indeed, increased levels of growth factors such as platelet-derived growth factor, eicosanoids such as hydroxyeicosatetraenoic acids, and oxidants such as oxidized low density lipoprotein have been reported in atheromatous arteries compared with normal arteries (3)(4)(5)(6)(7)(8)(9). As these molecules utilize divergent early mitogenic signaling events in the induction of VSMC growth (10,11), targeting inhibition of the activity of a single mitogen might not be able to suppress VSMC growth and lesion formation. However, identifying the mechanisms that are less redundant and that are involved in the mitogenic and chemotactic activities of many of these molecules may advance therapeutic developments that mitigate VSMC growth and vessel wall lesions.
Arachidonic acid, a polyunsaturated fatty acid, is an important component of membrane phospholipids and is released acutely in response to a variety of agonists, including growth factors, cytokines, hormones, and oxidants (12)(13)(14)(15)(16). Upon release, it is either metabolized via the cyclooxygenase, lipoxygenase, or cytochrome P450 monooxygenase pathway, producing prostaglandins, hydroperoxyeicosatetraenoic acids, or epoxyeicosatrienoic acids, respectively, or is reincorporated into membrane phospholipids via esterification involving arachidonoyl-CoA synthase and arachidonoyl lysophospholipid transferase (12,17). Arachidonic acid and its oxygenative metabolites, known as eicosanoids, are involved in the regulation of a variety of biological processes, including vascular tone (17,18). In addition, these lipid molecules have been reported to mediate intracellular signaling events in response to a number of stimuli (19 -24). A large body of data also suggest that arachidonic acid and its eicosanoid metabolites play an important role in cell survival and growth (25)(26)(27)(28)(29). Among the members of the large phospholipase A 2 family characterized thus far, cytosolic phospholipase A 2 (cPLA 2 ) appears to be the major source of eicosanoid production in response to some agonists in certain cell types (30 -32).
Janus-activated kinases (Jak) are a group of non-receptor tyrosine kinases that, via phosphorylation, modulate the activities of a group of transcription factors, viz. signal transducers and activators of transcription (STAT) (33,34). STAT proteins have been reported to be involved in the regulation of cell growth and differentiation (35)(36)(37)(38). To understand the molecular events of platelet-derived growth factor-BB (PDGF-BB)induced growth and survival in VSMC, we have studied the role of cPLA 2 and the Jak/STAT pathway. Here, we report for the first time that PDGF-BB induces the expression of cPLA 2 in VSMC in a sustained manner and that this response requires Jak-2-dependent activation of STAT-3. In addition, we show that Jak-2/STAT-3-dependent induction of expression of cPLA 2 is required for PDGF-BB-induced arachidonic acid release and growth in VSMC.
[ DNA Synthesis-VSMC with and without appropriate treatments were pulse-labeled with 1 Ci/ml [ 3 H]thymidine for the indicated times. After labeling, cells were washed with cold phosphate-buffered saline, trypsinized, and collected by centrifugation. The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysate mixture was passed through a Whatman GF/C glass-fiber filter. The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The filter was dried and placed in a liquid scintillation vial containing the scintillant fluid, and the radioactivity was measured in a Beckman LS 5000TA liquid scintillation counter.
Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts were prepared from treated or untreated VSMC as described previously (39). The protein content of the nuclear extracts was determined using a Micro BCA TM protein assay reagent kit (Pierce). Protein⅐DNA complexes were formed by incubating 5 g of nuclear protein in a total volume of 20 l consisting of 15 mM HEPES (pH 7.9), 3 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 g of bovine serum albumin, 2 g of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32 P-labeled oligonucleotide probe for 30 min on ice. In some experiments, nuclear extracts were preincubated with anti-STAT-3 antibodies for 3 h prior to protein-DNA binding assay. The protein⅐DNA complexes were resolved by electrophoresis on a 4% polyacrylamide gel using 1ϫ Tris/glycine/EDTA buffer (25 mM Tris-HCl (pH 8.5), 200 mM glycine, and 0.1 mM EDTA). Double-stranded oligonucleotides were labeled with [␥-32 P]ATP using the T4 polynucleotide kinase kit (Invitrogen) following the supplier's protocol.
Western Blot Analysis-After appropriate treatments, VSMC were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 l of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 100 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM sodium orthovanadate) and scraped into 1.5-ml Eppendorf tubes. After standing on ice for 20 min, the cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell lysates containing an equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels.

FIG. 1. PDGF-BB induces the expression of cPLA 2 and its activity in VSMC.
Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for the indicated times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed by Western blotting for cPLA 2 using its specific antibodies (A) or assayed for cPLA 2 activity using a commercially available kit (B). For a lane loading control, the same blot in A was reprobed with anti-STAT-3 antibodies. *, p Ͻ 0.01 versus control. PC, phosphatidylcholine. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Biosciences). After blocking in 10 mM Tris-HCl (pH 8.0) containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).
Transient Transfection and Chloramphenicol Acetyltransferase (CAT) Assay-VSMC were plated evenly onto 100-mm dishes and grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. At 50 -80% confluence, the medium was replaced with DMEM containing 0.1% calf serum, and cells were transfected with the pSIE-CAT plasmid using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. Thirty hours after transfection, VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for 4 h, and cell extracts were prepared. VSMC lysates were normalized for protein and assayed for CAT activity using [ 14 C]chloramphenicol and acetyl coenzyme A as substrates. In parallel experiments, VSMC were plated evenly onto 60-mm dishes and grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cells at 90% confluence were transfected with dominant-negative STAT-3 mutant plasmid (pFS3DM) DNA (5 g/60-mm dish) using Li-pofectAMINE Plus reagent. Cells were washed with phosphate-buffered saline 16 3. A, PDGF-BB stimulates tyrosine phosphorylation of Jak-2 and STAT-3 in a time-dependent manner in VSMC. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for the indicated times, and cell extracts were prepared. An equal amount of protein (40 g) from the control and each treatment was analyzed by Western blotting for phospho-Jak-2 (pJak-2) and phospho-STAT-3 (pSTAT-3) using their phospho-specific antibodies. B, PDGF-BB-stimulated tyrosine phosphorylation of STAT-3 is sensitive to inhibition by the Jak-2 inhibitor AG490. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for 30 min, and cell extracts were prepared. An equal amount of protein (40 g) from the control and each treatment was analyzed by Western blotting for phospho-STAT-3 using its phospho-specific antibodies. As a loading control, the blots were reprobed with anti-STAT-3 antibodies. In the case of PDGFR tyrosine phosphorylation analysis, an equal amount of protein from the control and each treatment was immunoprecipitated with anti-PDGFR antibodies, and the resulting immunocomplexes were analyzed by Western blotting using antibody PY20. pPDGF-R, phospho-PDGFR FIG. 4. AG490, a potent inhibitor of Jak-2, reduces PDGF-BBinduced translocation of tyrosine-phosphorylated STAT-3 from the cytoplasm to the nucleus. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for 30 min, and cytoplasmic and nuclear extracts were prepared. An equal amount of protein (40 g) from the cytoplasmic and nuclear extracts of the control and PDGF-BB-treated cells was analyzed by Western blotting for phospho-STAT-3 (pSTAT-3) using its phosphospecific antibodies. As a loading control, the blot was reprobed with anti-STAT-3 antibodies.

FIG. 5. AG490 reduces PDGF-BB-induced STAT-3 DNA-binding activity and STAT-3-dependent reporter gene expression in VSMC.
A, growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for 2 h, and nuclear extracts were prepared. Five micrograms of nuclear protein from the control and each treatment were incubated with 100,000 cpm of 32 P-labeled STAT-3 consensus oligonucleotide probe, and the protein⅐DNA complexes were separated by EMSA and subjected to autoradiography. In some protein-DNA binding reactions, nuclear extracts were preincubated with anti-STAT-3 antibodies (Ab) for 3 h. B, VSMC that were transfected with a STAT-3-dependent reporter plasmid (pSIE-CAT) were quiesced and treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for 4 h, and cell extracts were prepared. Cell extracts containing an equal amount of protein from the control and each treatment were analyzed for CAT activity using [ 14 C]chloramphenicol and acetyl coenzyme A as substrates. The substrate and products were extracted with ethyl acetate, separated by TLC, and subjected to autoradiography. 24 h of the 36-h treatment period as described above.
Statistics-All experiments were repeated three times with similar results. Data on AA release, cPLA 2 activity, and DNA synthesis are presented as means Ϯ S.D. The treatment effects were analyzed by Student's t test. p values Ͻ0.05 were considered to be statistically significant. In the case of CAT activity, EMSA, and Western blot analyses, one representative set of data is shown.

RESULTS AND DISCUSSION
To understand the mitogenic signaling events of PDGF-BB in VSMC, we have studied the role of cPLA 2 . Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. Equal amounts of protein from control and PDGF-BB-treated cells were analyzed by Western blotting for cPLA 2 using its specific antibodies. PDGF-BB induced cPLA 2 expression in a time-dependent manner, with 2-and 3-fold increases at 8 and 16 h of treatment, respectively (Fig. 1A). The increase in cPLA 2 expression induced by PDGF-BB also resulted in an increase in its activity as measured by hydrolysis of arachidonoyl thiophosphatidylcholine using a commercially available kit (Fig. 1B). Earlier studies from other laboratories have reported that cPLA 2 plays a role in AA release and growth in response to some agonists (15,40,41). To test the role of cPLA 2 in receptor tyrosine kinase agonist-induced AA release and growth, we studied the effect of arachidonyl trifluoromethyl ketone (AACOCF 3 ), a specific inhibitor cPLA 2 (42), on PDGF-BB-induced AA release and growth. Quiescent VSMC that were prelabeled with [ 3 H]AA were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AACOCF 3 (10 M) for 1 h, and [ 3 H]AA release was measured. PDGF-BB stimulated [ 3 H]AA release by ϳ6-fold, and this effect was significantly inhibited by AA-COCF 3 (Fig. 2A). To understand the role of cPLA 2 in PDGF-BB-induced growth, quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AACOCF 3 (10 M) for 24 h, and growth was measured by pulse labeling cells with 1 Ci/ml [ 3 H]thymidine for the last 12 h of the 24-h incubation period and determining the trichloroacetic acid-precipitable counts/min. PDGF-BB stimulated [ 3 H]thymidine incorporation by 9-fold, and this response was completely inhibited by AACOCF 3 (Fig. 2B). AACOCF 3 alone had no toxic effects in VSMC, at least for 72 h as determined by trypan blue dye exclusion assay.
To understand the signaling events underlying PDGF-BBinduced expression of cPLA 2 and AA release, we studied the role of the Jak/STAT pathway. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for various times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed by Western blotting for tyrosine phosphorylation of Jak-2 and STAT-3 using their phospho-specific antibodies. PDGF-BB stimulated tyrosine phosphorylation of both Jak-2 and STAT-3 in a time-dependent manner, with a maximum effect of 5-15-fold at 10 min and reaching basal levels by 4 h (Fig. 3A). Jak proteins phosphorylate STAT proteins at tyrosine residues and activate them, although other mechanisms were also reported to be involved in the activation of these transcription factors (43,44). To determine whether PDGF-BB-stimulated STAT-3 tyrosine phosphorylation is mediated by Jak-2, we tested the effect of AG490, a potent and specific inhibitor of Jak-2 (45). AG490 (25 M) significantly inhibited PDGF-BB-stimulated tyrosine phosphorylation of STAT-3 (Fig. 3B). The inhibition of PDGF-BBstimulated tyrosine phosphorylation of STAT-3 by AG490 was not due to its toxic effects in VSMC, as this compound did not FIG. 6. A and B, AG490 inhibits PDGF-BB-induced expression of cPLA 2 and its activity. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of AG490 (25 M) for the indicated times, and cell extracts were prepared. An equal amount of protein from the control and each treatment was analyzed either for cPLA 2 expression by Western blotting using its specific antibodies (A) or for cPLA 2 activity using a commercially available kit (B). PC, phosphatidylcholine. C, the dominant-negative STAT-3 mutant inhibits PDGF-BB-induced expression of cPLA 2 . VSMC were transfected with and without the dominant-negative STAT-3 mutant plasmid (pFS3DM), quiesced, and treated with and without PDGF-BB (20 ng/ml) for 16 h, and cell extracts were prepared and analyzed for cPLA 2 levels as described for A. *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus PDGF-BB treatment alone. affect the viability of these cells over a period of 72 h as measured by trypan blue dye exclusion assay. Because earlier studies have reported that PDGF-BB phosphorylates STAT-3 independent of Jak-2 and involving the PDGFR, we also tested whether AG490 affects PDGFR tyrosine phosphorylation. AG490 had no effect on tyrosine phosphorylation of PDGFR induced by PDGF-BB, a finding that suggests that the inhibitory effect of AG490 on STAT-3 tyrosine phosphorylation is specific.
Upon tyrosine phosphorylation, STAT proteins undergo either homo-or heterodimerization and translocate to the nucleus, where they bind (in this case, STAT-3) to their consensus DNA-binding sequence present in the promoter regions of genes and induce transcription (33,46). To test whether STAT-3, upon its tyrosine phosphorylation induced by PDGF-BB, translocates to the nucleus, quiescent cells were treated with and without PDGF-BB (20 ng/ml) for 30 min, and the cytoplasmic and nuclear extracts were prepared. An equal amount of protein from the cytoplasmic and nuclear extracts of control and PDGF-BB-treated cells was analyzed by Western blotting for the levels of tyrosine-phosphorylated STAT-3. Tyrosine-phosphorylated STAT-3 levels were detected only in the nuclear fraction of PDGF-BB-treated cells, and AG490 reduced these levels (Fig. 4). To determine whether translocation of tyrosine-phosphorylated STAT-3 correlates with increased transcription activation, STAT-3 DNA-binding activity was measured. Quiescent VSMC were treated with and without PDGF-BB (20 ng/ml) for 2 h, and nuclear extracts were prepared. An equal amount of nuclear protein from the control and each treatment was analyzed by EMSA for STAT-3 DNA-binding activity using 32 P-labeled STAT-3 consensus oligonucleotide as a probe. PDGF-BB increased STAT-3 DNA-binding activity by 6-fold, and it was inhibited by AG490 (Fig. 5A). Preincubation of nuclear extracts with anti-STAT-3 antibodies also significantly reduced PDGF-BB-induced protein⅐DNA complex formation (Fig. 5A). This result suggests that PDGF-BB-induced protein⅐DNA complexes contain STAT-3 either as homo-or heterodimers with other members of the STAT transcription factor family. To confirm that increased STAT-3 DNA-binding activity leads to increased transactivation activity, cells were transiently transfected with a STAT-3-dependent reporter plasmid (pSIE-CAT), quiesced, and treated with and without PDGF-BB (20 ng/ml) for 4 h, and cell extracts were prepared. Cell extracts normalized for protein were assayed for CAT activity. PDGF-BB induced STAT-3-dependent CAT activity by 4-fold, and AG490 substantially inhibited this response (Fig. 5B). To understand whether the Jak/STAT pathway plays a role in PDGF-BB-induced expression of cPLA 2 , we next studied the effect of AG490 on this event. AG490 completely inhibited PDGF-BB-induced cPLA 2 expression (Fig.  6A). Consistent with this observation, AG490 also inhibited PDGF-BB-induced cPLA 2 activity (Fig. 6B). To obtain additional evidence on the role of the Jak/STAT pathway in PDGF-BB-induced cPLA 2 expression, we tested the effect of a dominant-negative STAT-3 mutant, FS3DM (38). As shown in nuclear extracts were prepared. Five micrograms of nuclear protein from the control and each treatment were incubated with 100,000 cpm of 32 P-labeled STAT-3 consensus oligonucleotide probe, and the protein⅐DNA complexes were separated by EMSA. AG490 and/or forced expression of the dominant-negative STAT-3 mutant for 1 h, and [ 3 H]AA release was measured. Both AG490 and the dominant-negative STAT-3 mutant substantially reduced PDGF-BB-induced [ 3 H]AA release (Fig. 7A). To investigate whether the Jak/STAT-dependent induction of expression of cPLA 2 and AA release are required for PDGF-BB-induced VSMC growth, the effects of AG490 and the dominant-negative STAT-3 mutant on PDGF-BB-stimulated DNA synthesis were studied. As shown in Fig. 7B, PDGF-BB-induced DNA synthesis was significantly reduced by both AG490 and FS3DM. To validate the effects of FS3DM on PDGF-BBinduced cPLA 2 expression and DNA synthesis, its effect on endogenous STAT-3 DNA-binding activity was tested. Forced expression of FS3DM substantially reduced PDGF-BB-induced endogenous STAT-3⅐DNA complexes in VSMC (Fig. 7C).
The important findings of this study are as follows. 1) PDGF-BB, a receptor tyrosine kinase agonist and a potent VSMC mitogen, induced the expression of cPLA 2 in a sustained manner in VSMC. 2) PDGF-BB-induced expression of cPLA 2 also resulted in an increase in cPLA 2 activity. 3) AACOCF 3 , a selective inhibitor of cPLA 2 , attenuated PDGF-BB-induced AA release and growth in VSMC. 4) Jak-2-dependent STAT-3 activation mediated PDGF-BB-induced cPLA 2 expression and growth in VSMC. A number of PLA 2 enzymes, particularly group V secretory PLA 2 and group IV cPLA 2 enzymes, are involved in agonist-induced AA release (31,32,47,48). Interestingly, cross-activation between secretory PLA 2 and cPLA 2 enzymes has been observed in acute and delayed production of eicosanoids in many cell types, including human neutrophils and murine macrophages and mast cells (30,47,48). Acute activation of cPLA 2 in response to a number of agonists that are coupled to Ca 2ϩ mobilization, particularly G protein-coupled receptor agonists, cytokines, and phorbol esters, is mediated by the mitogen-activated protein kinase cascade and/or protein kinase C (49,50). In this study, we have shown for the first time that PDGF-BB stimulated sustained cPLA 2 activity via induction of its expression. Furthermore, the sustained expression and activity of cPLA 2 appeared to be mediated by and involved in Jak/STAT-dependent PDGF-BB-induced growth in VSMC. This conclusion is supported by the findings that AG490, a selective inhibitor of Jak-2, and a dominant-negative mutant of STAT-3 substantially reduced the expression and activity of cPLA 2 and DNA synthesis induced by PDGF-BB. Studies from other laboratories also indicate that cPLA 2 plays a role in serum-induced growth in human coronary artery smooth muscle cells (40). A potential role for the Jak/STAT pathway in the regulation of cell growth, differentiation, and survival activities in many cell types, including hematopoietic cells, has been demonstrated (35)(36)(37)(38)51). Based on these findings and the present observations, it is likely that cPLA 2 is one of the effector molecules that are involved in Jak/STAT signaling leading to induction of growth in VSMC by PDGF-BB. Some STAT transcription factors such as STAT-1 have also been reported to be involved in the induction of expression of cell cycle inhibitory molecules such as p21 waf1/cip1 and pro-apoptotic enzymes such as caspase-1 and thereby in apoptosis (52,53). In this regard, it is noteworthy that cPLA 2 -dependent AA release mediates oxidant-induced apoptosis in some cell types (54). In view of these findings, it can be speculated that cPLA 2 is distal in the path of Jak/STAT signaling to cell proliferation and/or apoptosis. Future studies are required to test whether the responsiveness of cPLA 2 to various agonists of cell growth and apoptosis is dependent on activation of different members of the STAT transcription factor family.
In summary, we have reported for the first time that the receptor tyrosine kinase agonist PDGF-BB induces the expres-sion of cPLA 2 in a manner that is dependent on activation of the Jak/STAT pathway. In addition, we have shown that Jak/ STAT-dependent induction of expression of cPLA 2 and AA release are involved in PDGF-BB-induced growth in VSMC.